Members of the TNFR (Tumor Necrosis Factor Receptor) superfamily play important roles in the induction of diverse signals leading to cell growth, activation and apoptosis. Smith et al., Cell, 76:959-962 (1994). Whether the signals induced by a given receptor leads to a cell's activation or death is highly cell-type specific and tightly regulated during differentiation of the cell. For example, the TNFRs can exert co-stimulatory signals for proliferation of naive lymphocytes, but can also induce death signals required for deletion of activated T lymphocytes. Smith et al., Cell, Id.
The cytoplasmic domains of these receptors lack intrinsic catalytic activity and generally exhibit no significant homology to each other or to other known proteins. Exceptions to this include Fas(CD95) and TNF-R1 which share significant homology within an 80 amino acid region of their cytoplasmic tails, referred to in the art as the “death domain.” Tartaglia et al., Cell, 74:845-853 (1993); Itoh et al., J. Biol. Chem. 268:10932-10937 (1993). Therefore, the TNFR family members are-believed to initiate different signal transduction pathways by recruiting different types of intra-cellular signal transducers to the receptor complex. Smith et al., Cell, Id.
Several types of intracellular signal transducers have been identified that initiate distinct signal transduction pathways when recruited to the members of TNFR superfamily. Rothe et al., Cell, 78:681-692 (1994); Cheng et al., Science, 267:1494-1498 (1995); Nakano et al., J. of Biol. Chem., 271: 14661-14664 (1996). Recent biochemical and molecular studies show that a class of signal transducing molecules are recruited to Fas(CD95) or TNFR1 via interaction of the death domains. Chinnaiyan et al., Cell, 81:505-512 (1995); Boldin et al., J. Biol. Chem., 270:7795-7798 (1995); Hsu et al., Cell, 81:495-504, (1995). For example, Fas(CD95) and TNFR1 recruit FADD(MORT1)/RIP or TRADD/FADD(MORT1)/RIP through the interactions of their respective death domains. Itoh et al., J. Biol. Chem., Id.; Tartaglia et al., Cell, Id.; Hsu et al., Immunity, 4:387-396 (1996). The clustering of these signal transducers leads to the recruitment of FLICE/MACH, and subsequently, to cell death. Muzio et al., Cell, 85:817-827 (1996); Boldin et al., Cell, 81: 803-815 (1996).
The TNFR family members can also recruit a second class of signal transducers called TRAF (Tumor necrosis factor Receptor Associated Factor), some of which are responsible for the activation of NF-kB or JNK. Hsu et al., Cell, 84:299-308 (1996); Liu et al., Cell, 87:565-576. TRAF proteins were identified by their biochemical ability to interact with TNFR2, CD40, CD30 or LT-βR receptors which interact directly with TRAFs via a short stretch of amino acids within their cytoplasmic tails but which do not interact with the death domain containing proteins. Sato et al., FEBS Letters, 358:113-118 (1995); Song et al., Biochem. J., 809:825-829 (1995); Lee et al., J. Exp. Med., 183:669-674 (1996).
Distinct members of the TRAF family have been identified as signaling components of the TNFR family. All TRAF members contain a conserved TRAF domain, approximately 230 amino acids in length, that is used for either homo- or hetero-oligomerization among the TRAF family to interact with the cytoplasmic regions of the TNFRs or for interactions with downstream signal transducers. Rothe et al., Proc. Natl. Acad. Sci. USA, 93:8241-8246 (1996); Song et al., Proc. Natl. Acad. Sci. USA, 93:6721-6725 (1996); Cheng et al., Genes Dev., 10:963-973 (1996). In addition to the TRAF domain, most of the TRAF family members contain an N-terminal RING finger and several zinc finger structures which appear to be important for their effector functions. Regnier et al., J. of Biol. Chem., 270:25715-25721 (1995); Hu et al., J. Biol. Chem., 269:30069-30072 (1994); Moisalos et al., Cell, 80: 389-399 (1995).
Several effector functions of TRAFs were revealed by recent experiments based on a transfection system. TRAF2, first identified by its interaction with TNFR2, was subsequently shown to mediate NF-kB activation induced by two TNF receptors, CD40 and CD30. Rothe et al., Science, 269:1424-1427 (1995); Lee et al., Proc. Natl. Acad. Sci. USA, 93:9699-9703 (1996). TRAF5 was also implicated in NF-kB activation mediated by LT-bR, whereas TRAF3 (also known as CRAF1, CD40 bp or LAP1) was shown to be involved in the regulation of CD40-mediated CD23 up-regulation in B cells. Cheng et al., Science, Id. Other TRAF members in the TNFR family-mediated signal transduction have potential effector functions as adapter proteins to recruit different downstream signal transducers to the receptor complex. For example, TRAF1 is required for the recruitment of members of the c-IAP (cellular Inhibitor of Apoptosis Protein) family to the TNFR2 signaling complex. Rothe et al., Cell, 83:1243-1252 (1995).
In addition to signal transduction with TNFR family members, TRAFs also have the potential to regulate other receptor-mediated signaling pathways. For example, TRAF6 is a component of interleukin-1 receptor (IL-1R) signaling complex in which it mediates the activation of NF-kB by IL-1R. Cao et al., Nature, 383:443-446 (1996). Since TRAFs form homo- or hetero-oligomers, it is suggested that the repertoire of TRAF members in a given cell type may differentially affect the intracellular signals triggered by these receptors. This may be accomplished by the selective interaction of TRAFs with a specific set of downstream signal transducers.
Although many aspects of TRAF-mediated effector functions leading to cellular activation have been defined, there is a need in the art for a determination as to whether TRAF proteins will also mediate the apoptotic signals induced by the death-domain-less members of the TNFR superfamily. Zheng et al., Nature, 377:348-351 (1995); Gruss et al., Blood, 83: 2045-2056 (1994); Amakawa et al., Cell, 84:551-562 (1996).
In view therefore, the present disclosure describes the isolation and characterization of a novel protein component that associates with the receptor-TRAF signaling complex and inhibits the TRAF2-mediated NF-kB activation, which can determine whether a given cell proliferates or dies.